Neisseria gonorrhoeae (Ng) is the causative agent of the sexually transmitted infection (STI), gonorrhea. Gonococcal infection has become resistant to almost every antibiotic that has been used. Ng has achieved superbug status and represents a global public health problem. Novel therapies against Ng are urgently needed. Ng lipooligosaccharide (LOS) modulates several aspects of virulence. Ng possess a LOS sialyltransferase (Lst) enzyme that scavenges CytidineMonophospho-N-acetylneuraminic acid (CMP-Neu5Ac; Neu5Ac is a nonulosonate [NulO] that belongs to the `sialic acid' family) from the host to sialylate its LOS. Two glycan extensions from heptose I of LOS can be sialylated: i) the lacto-N-neotetraose structure and ii) the less frequently encountered PK-like LOS. LOS Neu5Ac confers on Ng the ability to evade complement (C'), cationic antimicrobial peptides and neutrophils. Prior studies have shown that there is strong pressure in vivo for Ng to express `sialylatable' LOS. We have shown that Ng Lst is promiscuous and can `cap' Ng LOS with analogs of neuraminic and legionaminic acids. Of the five CMP-NulOs that capped LOS, incorporation of Neu5Ac9Az (Az [azido]) and Leg5Ac7Ac (Leg) not only failed to confer on Ng the ability to resist C', but also prevented the `physiologic' Neu5Ac from exerting its C' inhibitoy effects. Intravaginal treatment of mice with CMP- Leg5Ac7Ac significantly attenuated Ng infection. These exciting data show that disabling the physiologic `Ng sialic acid machinery' cripples its virulence. This proposal aims to advance the development of CMP-NulOs as a topical prophylactic against multidrug-resistant Ng. In Aim 1 we will test the efficacy in vivo of CMP- Leg5Ac7Ac against i) additional contemporary drug-resistant Ng and ii) Ng that express the `alternative sialylatable' (PK-like) LOS in the mouse vaginal colonization model. In Aim 2 we will examine whether CMP- NulO-treated cells express NulOs, which could be recognized as `foreign' by Ab and result in tissue damage. In the second (R33) phase of this proposal, CMP-Leg5Ac7Ac dose optimization studies will be performed in Aim 3. In Aim 4 we will define the arm(s) of the immune system that facilitates clearance of infection by CMP- Leg5Ac7Ac, using mice deficient in C3, cationic peptides (CRAMP KO), PMN-depleted or Rag 1 KO mice. The effects of NulO incorporation by Ng LOS on the interaction of Ng with relevant human Siglecs will also be examined. Aim 5 will examine the efficacy of CMP-NulOs in the context of chlamydia co-infection, a common clinical occurrence. Chlamydia increases Ng disease burden in mice and also enhances the likelihood of women acquiring Ng. Ng infection is host-restricted. Ng binds to the C' inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human specific manner, which results in selective resistance of human complement by Ng. Further, unlike all other mammals that possess both Neu5Ac and Neu5Gc, humans possess only Neu5Ac. In Aim 6, we will use human FH/C4BP double transgenic mice, and CMP-Neu5Ac hydroxylase KO mice (make only Neu5Ac, similar to humans) to test the efficacy of CMP-NulOs in a more human-specific context.